Glucagon-like peptide-1 (“GLP-1”) is a peptide hormone secreted by intestinal cells that has been shown in multiple studies to produce an enhancing effect on insulin secretion. Such studies have also shown that GLP-1 has more advantages than insulin in the treatment of type II diabetes mellitus. Most notably, GLP-1 is observed to be capable of enhancing β-cell division and therefore increasing β-cell counts, effects which have not been found in other medicines used for the treatment of diabetes. In addition, GLP-1 is effective in those patients who do not respond to treatment by the administration of sulfonylurea. Furthermore, because administration of GLP-1 does not enhance insulin secretion when the concentration of blood glucose is restored to normal levels, treatment with GLP-1 does not result in hypoglycemia. Therefore, in light of these and other data, GLP-1 is regarded as a desirable medicine to treat diabetes mellitus.
Exendin-4 is a 39 amino acid C-terminal amidated peptide analog of GLP-1 found in the venom of the Gila Monster (Heloderma horridum), with a 53% amino acid sequence homology to the GLP-1 peptide sequence. See, e.g., Eng, J., et al. “Isolation and Characterization of Exendin-4, and Exendin-3 Analogue from Heloderma suspectum Venom,” J. Bio. Chem., 267:11, p. 7402-7405 (1992), Young, A. A., et al., “Glucose-Lowering and Insulin-Sensitizing Actions of Exendin-4,” Diabetes, Vol. 48, p. 1026-1034, May, 1999. In terms of its activity, exendin-4 is a highly specific agonist for the GLP-1 receptor, and, like GLP-1, is able to stimulate insulin secretion. Therefore, like GLP-1, exendin-4 is regarded as an insulinotropic peptide.
However, unlike GLP-1, exendin-4 has a relatively long half-life in humans, because of its resistance to the dipeptidyl peptidase IV which rapidly degrades the GLP-1 sequence in vivo. Furthermore, it has been shown that, as compared to GLP-1, exendin-4 has a stronger capability to stimulate insulin secretion, and that a lower concentration of exendin-4 may be used to obtain such stimulating activity. See, e.g., U.S. Pat. No. 5,424,286, herein incorporated by reference. Therefore exendin-4 peptides or derivatives thereof (for examples of such derivatives see, e.g., U.S. Pat. No. 6,528,486, herein incorporated by reference, and its corresponding international application WO 01/04156) have a greater potential utility for the treatment of conditions involving the dysregulation of insulin levels (e.g., conditions such as diabetes) than either insulin or GLP-1.
In the present invention, several novel exendin-4 derivative sequences are disclosed which have been found to exhibit significant blood-glucose regulatory effects, including the ability to regulate blood glucose levels for long durations (i.e., long half-life) without hypoglycemic effects. In addition, the present invention discloses a novel recombinant method of producing such exendin-4 derivative sequences by producing these sequences as fusion proteins, which are then cleaved with the appropriate reagent to yield separate copies of the desired peptide sequence. In one aspect of this method, a novel procedure for producing exendin-4 fusion proteins that are cleaved by trypsin is disclosed.